Artificial three-dimensional microenvironment niche culture and methods of using the same

ABSTRACT

Described here are three-dimensional microenvironment niches prepared from biomaterial compositions that support growth and self renewal of stem cells. The invention also provides methods for inducing pluripotency in a somatic cell using chemical compounds, as well as methods for screening for compounds that can induce pluripotency in a somatic cell.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/636,548, filed Jun. 28, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/696,823, filed Apr. 27, 2015, now U.S. Pat. No.9,725,700, which is a continuation of U.S. patent application Ser. No.12/637,738, filed Dec. 14, 2009, now U.S. Pat. No. 9,045,737, whichclaims the benefit of priority to U.S. Provisional Applications Numbered61/122,360, filed Dec. 13, 2008, and 61/251,046, filed Oct. 13, 2009,the entire disclosures of which are incorporated herein by reference.

BACKGROUND

Induction of pluripotent status in somatic cells by directedreprogramming in-vitro, (induced pluripotent stem (iPS) cells) offersgreat potential for the generation of disease- and patient-specific celllines and cell therapy. Particularly, iPS cells provide the basis forpractical generation of patient-specific cells for customizedtransplantation. Generation of iPS cells from mouse and human cellsstarting from somatic fibroblasts has recently been reported.

Two independent groups have identified laboratory protocols to induceiPS cell reprogramming. Takahasi et al. have reported that mouse andhuman skin cells can be transformed into ES-like cells by transductionof four genes: OCT3/4, SOX2, KLF4, and c-MYC (Cell. (2007) 131:861-72).Subsequent report demonstrated generation without c-MYC, making theprocedure less prone to side effects such as induction of malignancy inhost animal models (see Nakagawa et al., Nat Biotechnol. (2008)26:101-6). A slightly different set of genes (OCT3, SOX2, NANOG andLIN28) has also been reported to reprogram human iPS cells. Such methodstypically utilize selectable markers (e.g., neomycin resistance markers)to isolate iPS cells. However, alternative procedures that eliminatedrug selection make such procedure more amenable to clinicalapplications in humans (Meissner et al. (2007) Nat. Biotechnol. 25:1177-81.).

Although promising, iPS techniques have several shortcomings that limitapplication of this approach for use in the clinic, including: thepotential of retroviruses to cause tumors in tissues derived from hostiPS cells; low efficiency of induction (approximately 1 in 5000-10000cells); the length of time the process requires (at least 20-24 days togenerate and identify iPS cells); and the need to use drug resistanceselection of iPS cells.

Thus, there remains an unmet need for patient-customized cell lines forcell therapy and tissue regeneration that are safe, can be rapidlyprepared and identified in quantity without the use of antibiotics orother drug-based selection.

Ongoing basic and applied research in this field continues to elucidateimportant findings about the pluripotency status as well as means ofinduction of the “iPS” status. For instance, genome-wide analysis of twokey histone modifications in iPS cells has indicated that iPS cells arehighly similar to ES cells. In addition, it has been reported thattranscription factor-induced reprogramming leads to the global reversionof the somatic epigenome into an ES-like state (Maheralli et al. (2007)Cell Stem Cell 1:55-70). iPS gene expression has been reported to berequired for about 10 days, after which cells enter a self-sustainingpluripotent state suggesting that factor-induced reprogramming is agradual process with defined intermediate cell populations that containcells poised to become iPS cells (Stadtfeld et al. (2008) Cell StemCell. 2:230-40).

Two overlapping groups of pluripotency-associated transcription factorshave been identified. The first group includes Nanog, Oct4, Sox2, Smad1and Stat3. The second, smaller group includes c-Myc (an oncogene thatboosts reprogramming efficiency), n-Myc, Zfx and E2f1. This may explainthe characteristic cooperative function of pluripotency-promoting genesand the need to have a number of the key genes unregulated in iPS cells(Chen et. al. (2008) Cell 133: 1106-17). Finally, using a combinedchemical and genetics approach for the generation of iPS cells,conditions that can potentially reduce the need for viral transductionof oncogenic transcription factors have been identified using neuralprogenitors and small molecules (Shi et al. (2008) Cell Stem Cell.2:525-528.).

To date reprogrammed iPS cells have been generated from a variety ofhost cells including hematopoietic, hair follicular, dermal fibroblasts,neuronal cells, umbilical cord blood cells, adult ocular progenitorcells, and pancreatic islet progenitor cells. iPS cells have also beengenerated from host cells of a variety of animals including human,mouse, rat, monkey, cow, sheep, goat, pig, horse, dog, cat, rabbit, andchicken. Moreover, pluripotent stem cells such as iPS and hESC have beendifferentiated into a variety of cell types including heart muscle,liver, neuronal, hematopoietic, pancreatic, bone, skin, sperm andretinal pigment epithelial cells, and in at least one case, a completeanimal has been generated with contributions from iPS cells.

Despite significant ongoing R&D efforts, the current unmet need forpatient-customized cell lines that could be used for cell therapy andtissue regeneration is the main driver for development of alternativemore practical iPS procedures, speeding the development of this earlystage discovery phase procedure into the clinic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the scores of cell growth, survival and overallculture health on the various polymers listed in Table 1.

FIG. 2 is a plot of the scores of cell growth, survival and overallculture health in the presence of the various extracellular matrixfactors listed in Table 2.

FIG. 3 is a plot of the scores of cell growth, survival and overallculture health in the presence of the growth factors listed in Table 3.

FIG. 4 is a plot of the scores of cell growth, survival and overallculture health in the presence of the crude factor extracts listed inTable 4.

FIG. 5 is a plot of the scores of cell growth, survival and overallculture health for the combinatorial subtractive screening conditionslisted in Table 5.

FIG. 6 shows the results of a preliminary ViPS pluripotency factorreplacement experiment described in EXAMPLE 3. OCT4, Sox2, Nanog, andKLF4 refer to exogenous genes introduced into HDF cells usinglentiviruses. BIO, PGE2, VA, and PGD2 refer chemical inducers(6-bromoindirubin-3′-oxime, prostaglandin E2, valproic acid, andprostaglandin J2, respectively that were used to replace one, two, threeor all ViPS pluripotency factors.

FIG. 7 shows the results of a screen of compounds for activation of thereporter constructs listed in Table 6 in HDF cells in a pluripotent stemcell microenvironment niche culture of the invention. Units shownrepresent relative fluorescence units from a green fluorescent proteinreporter. Compounds tested are listed in Table 8.

FIG. 8 shows the results of promoter-GFP expression in cells treatedwith combinations of virally introduced pluripotency factors andchemical inducers of pluripotency as described in EXAMPLE 6.

FIG. 9 shows the results of RT-PCR of CiPS cells using constructscontaining the primers listed in Table 10.

FIG. 10 is a section of a teratoma generated from CiPS cells asdescribed in EXAMPLE 8.

FIG. 11 is representation of the methylation status of CpGs in thepromoter of the Nanog and OCT4 genes from CiPS cells, hES cells and HDFcells as described in EXAMPLE 8.

DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. It must be notedthat, as used herein and in the appended claims, the singular formsinclude plural referents; the use of “or” means “and/or” unless statedotherwise. Thus, for example, reference to “a cell” includes a pluralityof such cells and reference to “the agent” includes reference to one ormore agents and equivalents thereof known to those skilled in the art,and so forth. Moreover, it must be understood that the invention is notlimited to the particular embodiments described, as such may, of course,vary. Further, the terminology used to describe particular embodimentsis not intended to be limiting, since the scope of the present inventionwill be limited only by its claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Suitable methods and materialsare described below, however methods and materials similar or equivalentto those described herein can be used in the practice of the presentinvention. Thus, the materials, methods, and examples are illustrativeonly and not intended to be limiting. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, general cell and tissue culture, transfection (e.g.,electroporation, lipofection, etc.), and the like. Enzymatic reactionsand purification techniques may be performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures may be generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. See e.g.,Teratocarcinomas and Embryonic Stem Cells: A Practical Approach(Robertson ed., Oxford: IRL Press, (1987)); Culture of Human Stem Cells(Freshney et al., eds., John Wiley & Sons, Hoboken, N.J.); Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)); Current Protocols inMolecular Biology (eds. Ausubel, et al., Greene Publ. Assoc.,Wiley-Interscience, NY); Harlow and Lane, Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988))the entire contents of which are incorporated herein by reference forany purpose. Unless specific definitions are provided, the nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, cell biology, cell culture, analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those well known and commonly used in the art.Standard techniques may be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients

Definitions

As used herein, “biomaterial” refers to any natural or man-madematerial, that is composed of or is derived from, in whole or in part,living matter which performs, augments, or replaces a natural function,such as a polymer scaffolding perfused with cells or cell extracts.

“Cell-based therapy”, as used herein, refers to treatment in which cellsor derivatives or products thereof, are induced to supplement, replace,repair or treat diseased, damaged or destroyed cells or tissues.

“Cell culture” as used herein, refers to the growth of cells in vitro inan artificial medium for research or medical treatment.

As used herein “cell line” refers to cells of a particular type that canbe maintained and grown in culture. Cell lines typically are homogeneousand well characterized, and can be stored (e.g. cryopreserved) for longperiods of time. Certain cell lines may have a finite life span, whileothers may divide indefinitely.

“Cloning” refers to the generation of identical copies, e.g. of a regionof a DNA molecule, or to generate and isolate genetically identicalcopies of a cell, or organism. In reference to cells grown in a tissueculture dish, a “clone” refers to a line of cells that is geneticallyidentical to the original parent cell, that is produced by cell division(mitosis) of the original parent cell.

“Culture medium” as used herein, refers to a liquid that covers cells ina culture dish and contains nutrients to nourish and support the cells.Culture medium may include growth factors added to produce desiredchanges in the cells.

“Differentiation” as used herein, refers to process through which a stemcell loses its capacity for self-renewal and becomes a mature anddefinitive cell type, thereby acquiring the features of a specializedcell.

“Dedifferentiation” as used herein, refers to the process by which adifferentiated cell reverts to a less specialized precursor, progenitoror stem cell state.

“Embryonic stem cells” or “ESCs” are primitive (undifferentiated) cellsderived from the inner cell mass of an embryo (e.g. a human blastocyst)that are capable of dividing without differentiating for a prolongedperiod in culture, and, because they are pluripotent, can differentiateinto any cell or tissue type. Mouse ESCs or mESCs can be injected into amouse blastocyst and contribute to the formation of a mouse. Human ESCsor hESCs are not known to contribute to the formation of a human being.

“Embryonic stem cell line” refers to embryonic stem cells that have beencultured under in vitro conditions that allow proliferation withoutdifferentiation for months to years.

Human embryonic stem cell or hESC refers to a human pluripotent stemcell derived from the inner cell mass (ICM) of a human blastocyst.

“Epigenetic” refers to changes in phenotype and/or gene expression bymechanisms other than changes in the underlying DNA sequence.

“Gene” as used here, refers to a functional unit of heredity that is asegment or segments of nucleic acid found and directs synthesis of RNA,which typically leads to production of a protein. Genes include both RNAcoding regions or sequences and control sequences, such as promoterslocated outside of RNA coding regions.

“High throughput screening” refers to technology for screening thatemploys automation and robotics to conduct hundreds or thousands ofbiological assay experiments within a short period of time. Typically,high throughput screening (HTS) systems will use rectangular plastictrays containing 96, 384, 1536, or 3456 wells (or more in microfluidicsystems), where each well may hold a small amount of liquid samplecontaining cells. Automated liquid handling can add factors or compoundsto test the effect on the cells. HTS can be used (for example) to screenhundreds of thousands of chemical compounds as potential drugcandidates, or to identify factors that induce pluripotency ordifferentiation of a cell in culture.

Induced pluripotent stem cell” or “iPS cells” are a type of pluripotentstem cell, similar to an embryonic stem cell, formed by the induction ofexpression of certain embryonic genes in a somatic cell. The process ofgenerating iPS cells, referred to as “reprogramming”, can involveintroduction of one or more, (often a combination of three to four)genes for e.g. transcription factors, delivered by retroviruses, into asomatic cell. iPS cells generated by viral, particularly retroviral(e.g. lentiviral) introductions of exogenous genes are referred to as“virally induced pluripotent stem cells” or “ViPS cells”. iPS cells canalso be induced by exposing somatic cells to soluble factors, includingchemical compounds, biochemicals, polypeptides, carbohydrates, lipidsand similar agents, and environmental niches that contain polymermatrices and immobilized or macromolecule-bound compounds and agents,without introduction of exogenous genetic material into the cells. SuchiPS cells are referred to as “chemically induced pluripotent stem cells”or “CiPS cells”. iPS cells also include cells that have been induced topluripotency through a combination of the introduction of exogenousgenes into a cell through viral or other vectors, and the treatment ofcells with non-genetic compounds, agents, and environments.

“In vitro” as used herein refers to experiments, methods or processesthat are performed or occur in an artificial environment, such as alaboratory culture tube or dish.

“In vivo” refers to experiments methods or processes that are performedor occur within the body of an organism, such as in animals in humanclinical trials or treatments.

“Microenvironment” as used herein, refers to the molecules, includingsmall molecules (such as compounds and other soluble factors),macromolecules (such as insoluble polymers), nutrients, growth factors,fluids, growth factors, cytokines and parameters such as pH, ionicstrength and gas composition as well as adjacent cells, tissues, and thelike, surrounding a cell in an organism or in the laboratory.

“Niche” refers to a microenvironment in which a cell is situated that isadapted to the phenotypic characteristics of the cell. Thus a “stem cellniche” is a microenvironment surrounding a stem cell that enables thestem cell to self-renew by dividing and giving rise to identical progenycells. Changes in a stem cell niche may result in differentiation of thestem cell. Thus, there may be microenvironmental niches that promotedifferentiation of the stem cell into various cell lineages (i.e.ectoderm, mesoderm, and endoderm) as well as more specialized nichessuitable for the further differentiation and specialization of cellsinto each of the types of cells in an organism (e.g. nerves, muscle,blood cells and the like).

“Multipotent” refers to the ability to develop into a limited number ofcell types type of an organism. For example, hematopoietic stem cellsare multipotent cells that can produce the various cell types found inblood.

By contrast, “pluripotent” cells are those that have the ability to giverise to all of the various cell types of the body, but cannot give riseto extra-embryonic tissues such as the amnion, chorion, and othercomponents of the placenta, and cannot produce a living organism.Pluripotency can be demonstrated by providing evidence of stabledevelopmental potential, to form derivatives of all three embryonic germlayers from the progeny of a single cell and to generate a teratomaafter injection into an immunosuppressed mouse. Other indications ofpluripotency include expression of genes known to be expressed inpluripotent cells, characteristic morphology and patterns of genomic DNAmethylation known to be related to pluripotent epigenetic status.

“Totipotent” as used herein, refers to the ability of a cell, having theability to develop into all types of cell including extraembryonictissues (e.g. placenta) and to give rise to an entire organism (e.g. amouse or human).

“Pluripotency factors” refers to the genes and/or gene products requiredto induce and/or maintain the pluripotent state of a stem cell. Certainmethods for reprogramming involve introducing a combination of three tofour pluripotency factor genes into a somatic cell using retroviruses.More recently, reprogramming methods have been derived that employdifferent, overlapping sets of pluripotency factors. Reprogrammingmethods described herein have been developed that use non-geneticagents, referred to generally as “chemicals” “compounds” or“compositions”, that induce expression of endogenous pluripotencyfactors in a cell.

“Progenitor cell” is an early descendant of a stem cell that candifferentiate, but cannot self-renew itself anymore. Pluripotent stemcells can differentiate into more specialized, non-pluripotent stemcells, such as hematopoietic stem cells. Thus, early progenitor celldescendents of a pluripotent stem cell may become committed todifferentiate into hematopoietic lineages, including multipotenthematopoietic stem cells, and may therefore be referred to ashematopoietic progenitor cells.

“Proliferation” refers to the expansion in the number of cells bydivision (mitosis) of single cells into two daughter cells.

“Reprogramming” as used herein, refers to the process of changing orinducing a cell from a more differentiated state into a lessdifferentiated state. Inducing a differentiated somatic cell (e.g. adermal cell) to de-differentiate into a pluripotent stem cell (iPS), isaccomplished through the process of reprogramming.

“Self-renewal” refers to the ability of a stem cell to divide and formmore stem cells with identical properties to the parent stem cell,thereby allowing the population of stem cells to be replenishedindefinitely.

“Somatic cell” as used herein, refers to differentiated body cells.

“Stem cells” are cells that have the ability to divide, giving rise toidentical daughter cells (self-renewal) and progeny cells that candifferentiate into specialized cells that are different from the stemcell parent. Stem cells can be totipotent, pluripotent or multipotent.Totipotent stem cells include zygotes. Pluripotent stem cells include EScells derived from the inner cell mass of a blastocyst stage embryo,while multipotent stem cells include “adult stem cells” or “somatic stemcells” which are derived from the early embryos (post-blastocyst stage),fetus or adult.

“Undifferentiated” refers to a cell that has not yet developed into aspecialized cell type.

“Expression” or “gene expression” as used herein refers to theconversion of the information from a gene into a gene product. A geneproduct can be the direct transcriptional product of a gene (e.g., mRNA,tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other typeof RNA) or a protein produced by translation.

“Operably linked” as used herein, means without limitation, that thecoding region is in the correct location and orientation with respect tothe promoter such that expression of the gene will be effected when thepromoter is contacted with the appropriate polymerase and any requiredtranscription factors.

The present invention is based on the concept that the environment of acell, particularly the microenvironment immediately surround a cellinfluences its phenotype, and pluripotency or differentiation status.Thus, it was postulated that the more suitable an artificialmicroenvironment surrounding a cell could be for maintainingpluripotency, the more likely it would be that non-pluripotent cells inthe same environment could be induced to a pluripotent state, and themore easily such somatic cells could be reprogrammed.

It was generally believed by the inventor that non-genetic compounds,such as small organic molecules, could be suitable for replacement ofthe introduction of exogenous pluripotency factors by gene transduction,through a reprogramming process that involved inducing iPS signaltransduction pathways. The present invention provides methods foridentifying non-genetic activators, such as small organic moleculeactivators, compounds, drugs, hormones, growth factors and the like, ofiPS signal transduction pathways that can replace introduction ofexogenous pluripotency factors by gene transduction for the induction ofpluripotent status of somatic cells. The resulting cells are referred toas chemical iPS cells or CiPS.

In previous studies, the inventors developed and optimized an artificialin-vitro modeled biomaterial hydrogel microenvironment niche forhematopoietic stem cells (HSCs). The artificial microenvironment nichewas found to support HSC proliferation and self-renewal, whilemaintaining the ability of HSCs to differentiated into hematopoieticnon-stem cells. Moreover, it was discovered that the HSCmicroenvironment niche could be adapted to use as a self-renewalscreening assay for hematopoietic stem cells (CD3⁴⁺), through whichpotential activators of HSC self-renewal could be screened andidentified. In developing the HSC microenvironment niche, humanhematopoietic stem cells were cultured in a particular microenvironmentand then contacted with potential self-renewal activation andmaintenance factors, and self-renewal was measured as a means foroptimizing the microenvironment niche. Activators thus identified couldthen be tested for their effects on somatic cells.

The present invention is based on the extension of studies with HSCenvironmental niche culture, to develop microenvironmental nichescomprising biomaterial compositions optimized for pluripotent stem cells(i.e. that support growth and self renewal of pluripotent stem cells).It was reasoned that an optimized pluripotent stem cellmicroenvironmental niche could provide an appropriate environment inwhich to induce pluripotency of differentiated cells such as skin cells(i.e. dermal fibroblasts), thereby developing methods for inducingpluripotency and for identifying compounds that could inducepluripotency in a differentiated cell.

Among the teaching drawn upon to develop HSC microenvironmental nicheculture, was the observation that certain hydrogels, particularlyhyaluronan hydrogels, markedly improved the success of HSC growth andself-renewal, and facilitated screening efforts by permittinghigh-throughput screening in an array format.

Artificial In-Vitro Modeled Biomaterial Hydrogel Microenvironment Niche.

Hyaluronan hydrogels are remarkably versatile. They can be tailored tocovalently attach peptides, non-covalently incorporate proteins (ECMproteins and cytokines), and to have different rigidities (compliances).Growth factors are retained within and slowly released from the hydrogelover the course of several weeks in culture. Growth factors aretypically protected from proteolysis so that their bioactivity ismaintained in longer term cultures. Incorporating growth factors (e.g.cytokines) in the hydrogel instead of the media allows for a dramaticreduction in the quantity needed. In certain embodiments, hyaluronanhydrogel chemistry available from Glycosan, (Salt Lake City, Utah) canbe used. This is a xeno-free hydrogel containing thiol-modifiedhyaluronan and a polyethylene glycol diacrylate (PEGDA, MW 3400) crosslinker that can gelatinize in less than 20 minutes for cellencapsulation.

Cells can be recovered from hyaluronan hydrogels using a variety ofmethods. For example, cells plated on the surface of the hydrogel can berecovered using traditional protocols for trypsin, collagenase, dispaseand the like, including but not limited to, Accutase or TrypLE products.Hydrogel-encapsulated cells can be readily released using enzymaticdigestion with hyaluronidase (an enzyme that digests the large HApolymer), thus dissolving the hydrogels, without harming theencapsulated cells.

In one embodiment of the invention, the microenvironment niche is a cellmatrix array (CMA) adapted for rapid analytical identification andtesting of the experimental parameters for optimum and derivation ofCiPS cells. Such CMAs contain microspots of approximately 150 microndiameter containing factors contact printed (pin spotted) on a surfacepre-coated with a formulation of hyaluronan hydrogel. This formatfacilitates multiplexing and permits direct analysis of cells by, e.g.,ICC, staining for DAPI, alkaline phosphatase, and the like. Largerformats of the CMA are also contemplated for use in the methods of thepresent invention (e.g. 6-, 12-, 24-, 96- and 384-well plates, one wellper hyaluronan and factor composition) and are suitable for applicationsthat require larger yield of cells, such as for FACS (fluorescenceactivated cell sorting) analysis. In one aspect of the invention, CMA isperformed using hyaluronan hydrogel containing a cocktail of ECM factorsin mTeSR-1 media.

In developing the HSC microenvironment niche CMA assays for non-adherenthematopoietic stem cells, a method for preventing migration of the cellswas required. Thus, 3-D cultures were used in which cells were embeddedin the hyaluronan hydrogels. Although other cells, such as embryonicstem cells (ESCs), fibroblasts, and a vast array of differentiated cellscan be grown on the surface of a culture dish, hydrogel or other matrix(2-D culture), it was fortuitously discovered that improvements in themorphological appearance of ESCs and higher plating efficiency could beobtained when cells were grown in 3-D culture versus 2-D culture. Thus,although methods have been previously reported to support the growth andself-renewal of ESCs in culture, for example, by growing HSCs in thepresence of leukocyte inhibitory factor (LIF) and/or on layers of feedercells (see e.g. Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach (1987) Oxford University Press), and more recently that in theabsence of animal cells or cell products (see e.g. U.S. Pat. No.7,442,548; Ludwig et al., Nat. Methods. (2006) 3:637-46), the 3-Dhydrogel microenvironment niche culture compositions and methodsdescribed herein provide superior conditions for screening for chemicalinduction of pluripotency. While not wishing to be bound by a particulartheory, it is believed that a 3-D microenvironment which surrounds thecells with hydrogel polymer matrix and other biomaterials in connectionwith the hydrogel, more closely simulates the in vivo environment of apluripotent stem cell than does a 2-D culture environment.

Thus, in one embodiment, the invention provides a 3-D microenvironmentniche comprising a biomaterial composition that supports growth and selfrenewal of a stem cell, such as a pluripotent stem cell. In certainaspects of the invention, the stem cell is an embryonic stem cell suchas a human embryonic stem cell (hESC). However, it should be noted thatconditions provided by the three-dimensional microenvironment nicheculture are generally suitable for growing a wide variety of cell typesin which it is advantageous to surround the cell with hydrogelmatrix-associated biomaterials. Cells contemplated for growth in thethree-dimension microenvironment niche of the invention includepluripotent stem cells, adult stem cells (e.g., hematopoietic stemcells), and somatic cells. While the compositions of themicroenvironment niche described herein have been optimized for growthof human ES cells through the elimination of e.g. animal cells, it willbe well within the level of skill in the art to adapt the teachings ofthe present invention to culture of cells of other species, such asanimals, particularly mammals, including but not limited to non-humanprimate, rodent, canine, feline, ovine, porcine and equine cells.

In certain embodiments of the invention, the 3-D microenvironment nichecomprises a hyaluronan polymer. The optimized microenvironment nichecontaining this polymer was found superior to similar formulationscontaining Matrigel or more than a dozen other biopolymers. Thus, 3-Dmicroenvironment niche cultures containing hyaluronan are preferred.However, it is well within the level of skill in the art to test new oradditional polymers or polymer combinations for use in the 3-Dmicroenvironment niche cultures of the invention based on the teachingspresented below in EXAMPLE 1. There may be a reasonable expectation ofsuccess in substituting another biopolymer where the biopolymer hasproperties similar to hyaluronan, which is also known as a polymer ofhyaluronic acid or hyaluronate, and is an anionic, non-sulfatedglycosaminoglycan distributed widely throughout connective, epithelial,and neural tissues. Naturally occurring glycosaminoglycan polymers,particularly those found in extracellular matrices of human or non-humananimal tissues are contemplated within the scope of the invention.

The optimized microenvironment niche according to the invention alsocontains at least one component selected from laminin, fibronectin,vitronectin; epidermal growth factor (EGF); fibroblast growth factor(FGF); Noggin; SIS; and EHS basement membrane. In certain embodiments,laminin is present at a concentration of 0.5 to 500 μm/ml, typically 1to 100 μm/ml and most frequently about 5 μg/ml. In certain embodiments,fibronectin is present at a concentration of 0.5 to 500 μm/ml, typically1 to 100 μm/ml and most frequently about 5 μg/ml. In certainembodiments, vitronectin is present at a concentration of 0.6 to 600μg/ml, typically 1 to 100 μm/ml and most frequently about 6 μm/ml. Incertain embodiments, EGF basement membrane is present at a concentrationof 4 to 4000 ng/ml, typically 10 to 1000 ng/ml and most frequently about40 ng/ml. In certain embodiments, FGF is present at a concentration of20 to 220 ng/ml, typically 100 to 6000 ng/ml and most frequently about220 ng/ml. In certain embodiments, Noggin is present at a concentrationof 1.5 to 15000 μm/ml, typically 15 to 5000 μm/ml and most frequentlyabout 150 ng/ml. In further embodiments, SIS is present at aconcentration of 5 to 5000 μl/ml, typically 100 to 10000 ml and mostfrequently about 50 μg/ml. In yet further embodiments, EHS basementmembrane is present at a concentration of 5 to 5000 μm/ml, typically 100to 1000 μm/ml and most frequently about 50 μm/ml.

In one aspect of the invention, the microenvironment niche according tothe invention comprises about 5 μg/ml fibronectin, about 6 μm/mlvitronectin, about 40 ng/ml EGF, about 220 ng/ml FGF, about 150 ng/mlNoggin, about 50 μl/ml SIS and 50 μm/ml EHS basement membrane. Incertain embodiments of the invention, one or more or all of theaforementioned components is included in hydrogel formulation itself; inother embodiments, one or more or all of the aforementioned componentsis included in included in a soluble culture medium in which thehydrogel is bathed; in yet further embodiments, one or more or all ofthe aforementioned components is included in both the hydrogelformulation and is present in a soluble culture medium in which thehydrogel is bathed.

Suitable adaptations and variations of the microenvironment niche can bemade by the skilled artisan by following the teaching outlined inEXAMPLES 1 and 2. Particularly, providing a basic microenvironment nichebiomaterial composition described herein, adding or substitution one ormore additional biomaterials, nutrients, growth factors, polymers or thelike, and comparing the growth and self renewal of stem cells, such apluripotent stem cells, e.g. hESCs in the presence of the basicmicroenvironment niche biomaterial composition and in the modifiedmicroenvironment niche composition, scoring cell growth, survival andappropriate stem cell morphology, and selecting additions orsubstitutions that improve the score of cells grown in the modified 3Dmicroenvironment niche composition.

CMA-Based Screening for Induction of Pluripotency

The present invention also provides CMA-based drug screening using the3-D microenvironment niche biomaterial compositions described herein. Incertain aspects of the invention the CMA is be adapted to highthroughput screening. Compound screening can be performed within thisoptimal 3-D microenvironment niche biomaterial compositions assaybackground to accurately identify, select and quantify self renewal ofstem cells or pluripotency induction of somatic cells.

In one embodiment, the present invention provides a method of screeningfor compounds that induce pluripotency of a somatic cell, that includesthe steps of contacting a test compound with a somatic cell that is incontact with (i.e. surrounded by or embedded within) a three 3-Dmicroenvironment niche described herein; measuring expression of anendogenous pluripotency factor in the somatic cell; and selecting testcompounds that increase the expression of the endogenous pluripotencyfactor in the somatic cell. As described in greater detail below in theEXAMPLES, an increase in the expression of certain endogenouspluripotency factors in the somatic cell correlates to induction ofpluripotency. Endogenous pluripotency factors contemplated for use inscreening methods of the invention can be any endogenous gene, theexpression of which is correlated to a phenotype or property of apluripotent stem cell, such as an undifferentiated phenotype, cellgrowth and self-renewal, and an ability to differentiate into all threegerm layers. Such pluripotency factors include, but are not limited to,OCT3/4, SOX2, LIN28, KLF4, cMYC and NANOG. In addition, it may be ofbenefit to measure expression of an endogenous factors in the somaticcell that support or promote pathways known to be activated inpluripotent cells, such as the Wnt pathway. In yet further embodiments,expression of additional pluripotency factors or repression ofpluripotency-suppressive factors may be targeted and measured forspecific types of somatic cells. For example Takenaka et al. havereported ViPS generation from cord blood cells using OCT3/4, SOX2,Krüppel-like factor 4, c-MYC expressing lentiviruses in combination withp53 knockdown (see Exp Hematol. (2009) Nov. 14); and Li et al. (Nature(2009) 4601136-9) have reported increased efficiency of ViPS byincluding inhibition of Ink4/Arf in mouse and human cells.

In certain embodiments of the invention, compounds are preselected forscreening based on known or reported effects on abilities to affectexpression of pluripotency factors or pathways thought to be importantin regulating expression of one or more pluripotency factors orpromoting a phenotype or characteristic of a pluripotent stem cell, suchas growth, characteristic morphology, self-renewal, and ability todifferentiate. Thus, for example, chemical inducers of iPS cells canalso be pre-selected or screened based on their ability to trigger theViPS process, which has been shown to require exogenous expression of 3main iPS factors: Oct3/4, Sox-2, and Klf4. Thus, one embodiment thepresent invention provides methods for identifying CiPS activators byculturing cells in a biomaterial hydrogel microenvironment niche,contacting the cultured cells with potential activators, and measuringthe effect of potential activators on the expression of Oct3/4, Sox-2,and/or Klf4. Expression can be measured by any suitable means known inthe art. In one embodiment, expression is measured using constructs inwhich promoter regions of Oct3/4, Sox-2, and/or Klf4 are cloned upstreamof a green fluorescent protein or other reporter gene. In certainembodiments, the time course and kinetics of promoter-reporter geneexpression is compared to that which occurs during ViPS induction asdescribed in Takahashi. et al ((2007), Cell 131:1-12); Junying et al.((2007) Science 318: 1917-1920); Meissner et al. ((2007) NatureBiotechnology 25 (10): 1177-1181)); and Nakagawa et al. ((2007) NatureBiotechnolgy doi:10.1038/nbt1374)).

Using a pre-selection approach, a cocktail of four compounds wasdiscovered that could replace the expression of pluripotency factorsOCT3/4, SOX2, NANAOG, and KLF4 from exogenous polynucleotides virallyintroduced into human dermal fibroblasts, (see EXAMPLE 3, below).Further compounds were discovered upon screening a preselected libraryof compounds, that could improve or enhance the efficiency of inductionof pluripotency, as illustrated below (see e.g. FIGS. 7 and 8, andaccompanying text).

Of particular interest for pre-selection are: compounds that activateWnt pathway; compounds that activate the cyclooxygenase pathway and itscross talk with the Wnt pathway; compounds with histone deacetylaseactivity; compounds that increase OCT3/4 expression; compounds thatincrease Sox-2 expression; compounds that increase Klf4 expression;compounds that increase Nanog expression; and compounds that inhibit p53and related pathways.

Using methods of the invention, compound classes that influence signaltransduction pathways and play key roles in self renewal signaltransduction pathways were identified in a preselected librarycompounds. From approximately 1800 compounds, 7 compounds have beenidentified that together or in various combinations, can inducepluripotency of HDF cells using a CMA-based drug screening of theinvention with the 3-D microenvironment niche biomaterial compositionsdescribed herein. Of particular interest are compounds that stimulatemultiple pluripotency factors. For example, compound valproic acid wasfound to stimulate expression of both SOX2 and OCT4 promoters (see FIG.7). It may thus be possible to chemically induce pluripotency of somaticcells using only one or a limited number of chemical inducers.

In one aspect of the invention, libraries containing uncharacterizedcompounds, such as combinatorial libraries of compounds, can be screenedfor their ability to effect self-renewal by stimulating expression ofendogenous pluripotency factors. The screening methods of the inventioncan also be used in conjunction with structure assisted design,medicinal chemistry, and structure activity relationship analysis ofsmall molecule activator/inhibitors of self-renewal pathways, providingalternatives for compound structure availability.

Cells suitable for use in iPS trigger assays can be any cells, and arepreferably mammalian somatic cells, such as mouse or human somaticcells. In certain embodiments, the cells are fibroblasts, (e.g., humandermal fibroblasts (HDF)).

In certain embodiments of the invention CiPS cells are characterized atthe cellular and molecular level and compared to V iPS cells. In theseaspects, cells are grown and induced for CiPS in the 3-Dmicroenvironment niche biomaterial compositions of the invention.Control cells are induced using genetic (e.g., viral) transduction ofiPS cells based on established protocols that are known in the art.Samplings of cells at time=0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24days post treatment, scored for characteristic ES cell morphology, andvalidation of cell reprogramming. Cell reprogramming can be measured byany methods known in the art, including but not limited to, expressionof pluripotency associated genes using, for example, RT-PCR; immunoassayfor pluripotency biomarker expression; methylation analysis of promotersaffected by stem cell specific epigenetic events, such as the Oct4 andNanog promoter; and the ability to form teratomas in SCID mice, givingrise to all three embryonic germ layers.

In certain embodiments, cultures maintained for 24 days are analyzed forthe presence of iPS colonies by morphological identification. CiPS andViPS colonies (subclones) are then isolated, propagated and analyzed. Inaddition, the similarities and/or differences between chemically andgenetically induced iPS cells can be identified at the molecular andcellular level, and the rate and efficiency of iPS colony induction canbe compared. Other standard culture analyses such as routine cellviability testing using dye exclusion, are performed throughout themethods of the invention.

CMA methods of the invention permit rapid testing of a variety ofparameters, including various chemical activators, in a combinatorialmanner making possible comprehensive analysis of agonistic versusantagonistic, and “cross talk” effects (e.g. a growth factor cross talkwith an ECM factor or a rigidity feature).

Chemical Induction of Pluripotency in Somatic Cells

Using the overall scheme described above, an exemplary protocol forchemical induction was devised that resulted in CiPS inductionefficiencies equal to or greater than those observed with ViPS inparallel. Thus, the present invention also provides a method forinducing pluripotency of a somatic cell, that includes the steps ofproviding a somatic cell in contact with a 3-D microenvironment nichebiomaterial composition that supports growth and self-renewal of a stemcell (e.g. an embryonic stem cell, such as a hESC), and contacting thesomatic cell with at least one compound that induces expression of atleast one endogenous pluripotency factor. The somatic cell can be anynon-pluripotent cell, such as a somatic stem cell (e.g. Adult stemcells, HSCs) and somatic non-stem cells, such as a fibroblasts (e.g. adermal fibroblast), and can be from a cultured cell such as a cell lineor primary culture, or it can be a cell that has not previously beencultured, such as from a tissue or organ (e.g. a biopsy specimen) orcell from a bodily fluid, such as blood, urine, amniotic fluid or lymph.Contemplated for use in the present invention are cells from a varietyof organisms, particularly mammals, including but not limited to humanand non-human primates (e.g. monkey, chimpanzees, macaques, baboons andthe like) rodents (e.g. mice, rats, hamsters), canines, felines, ovines,porcines and equines.

In one embodiment of the invention, the cell is embedded in the 3-Dmicroenvironment niche by preparing a hydrogel matrix containing abiomaterial composition of the invention, and mixing the hydrogel withthe cell; and allowing the hydrogel to solidify. In this manner, thecell is completely surrounded by the microenvironment niche.

In certain aspects of the invention, the microenvironment niche includesa hyaluronan hydrogel polymer and about 5 μg/ml fibronectin, about 6μg/ml vitronectin, about 40 ng/ml EGF, about 220 ng/ml FGF, about 150ng/ml Noggin, about 50 μl/ml SIS and 50 μg/ml EHS basement membrane.

After plating, the cell may optionally be treated with Colcemid oranother cell synchronization agent, which has been found to improve CiPSinduction efficiency. The cell is then cultured in microenvironmentniche biomaterial composition for 1-14 days, typically 3-10 days andmost often for 7 days in the with additions and changes of fluid mediaas needed (approximately every 2 days).

iPS cell induction is accomplished by contacting the cell with at leastone compound that induces expression of at least one endogenouspluripotency factor, as described above. The at least one pluripotencyfactor can be OCT3/4, SOX2, LIN28, KLF4, cMYC or NANOG. In certainaspects of the invention, the at least one pluripotency factor activatesa Wnt pathway, activates a Cyclooxygenase pathway, or inhibits a p53activity. An exemplary combination of compounds includes at least one of6-bromoindirubin-3′-oxime (BIO); valproic acid; prostaglandin J2; andprostaglandin E2 and optionally); indirubin-5-nitro-3′-oxime (INO);1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanoneHBr (Pifithrin-α); and/or2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.

In certain embodiments, induction is accomplished by contacting the cellwith a combination of 6-bromoindirubin-3′-oxime (BIO);indirubin-5-nitro-3′-oxime (INO); valproic acid;2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine;1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanoneHBr (Pifithrin-α); prostaglandin J2; and prostaglandin E2. For example,the final composition of chemical inducers can include 0.1 to 100 μM,frequently 0.4 to 40 μM, and typically about 4 μM6-bromoindirubin-3′-oxime (BIO); 0.1 to 100 μM, frequently 0.4 to 40 μM,and typically about 4 μM indirubin-5-nitro-3′-oxime (INO); 0.05 to 50mM, frequently 0.2 to 20 mM, and typically about 2 mM valproic acid; 0.1to 1000 nM, frequently 2.5 to 250 nM and typically about 25 mM2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-aphthyridine; 0.1 to1000 μM, frequently 3 to 300 μM, and typically about 30 μM1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanoneHBr (Pifithrin-α); 0.5 to 500 μM, frequently 1 to 100 μM and typicallyabout 10 μM prostaglandin J2; and 0.5 to 500 μM, frequently 1 to 100 μMand typically about 10 μM prostaglandin E2.

In one embodiment, the induction is accomplished by the additionchemical inducers to a final concentration of about 4 μM6-bromoindirubin-3′-oxime (BIO); about 2 mM valproic acid; about 10 μMprostaglandin J2; about 10 μM prostaglandin E2; and optionally about 4μM indirubin-5-nitro-3′-oxime (INO); about 25 mM2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-aphthyridine; and/orabout 30 μM Pifithrin-α.

The induction is allowed to proceed for about 1-6 weeks, frequently 2-3weeks and most typically about 32 days with changes of the solublemedium and replenishment of chemical inducers as needed to maintain theviability of the cells (50% media change approximately every 2 days).

Following induction, cells are dissociated from the 3-D microenvironmentniche matrix and iPS cells generated thereby can be plated in either 3-Dor 2-D culture, (such as by plating on top rather than within themicroenvironment niche biomaterial composition of the invention), andwithout the presence of inducers. Notably, the CiPS methods of theinvention produce iPS cells with phenotypic characteristics of ViPScells and similar to hES cells (see EXAMPLE 8 below), without thetransfer of exogenous pluripotency factors to the cells, without the useof viral vectors and without continued, long-term presence of theinducing agent.

Chemically Induced iPS Cells (CiPS Cells)

In yet another embodiment, the present invention provides CiPS cellsinduced by a method of the invention. For example, the inventionincludes pluripotent stem cells induced by at least one or a combinationof 6-bromoindirubin-3′-oxime (BIO); valproic acid; prostaglandin J2;prostaglandin E2; and optionally indirubin-5-nitro-3′-oxime (INO);2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-aphthyridine; and/orPifithrin-α.

Chemically induced pluripotent cells of the invention have at least onecharacteristic of an ES cell or a reprogrammed, ViPS cell, such ascharacteristic morphology in culture, self-renewal, stable developmentalpotential to form derivatives of all three embryonic germ layers fromthe progeny of a single cell, ability to generate a teratoma afterinjection into SCID mouse giving rise to all three embryonic germlayers; patterns of genomic DNA methylation known to be related topluripotent epigenetic status; expression of pluripotency associatedgenes and presence of biomarkers of pluripotency.

EXAMPLES Example 1: Pluripotent 3D Culture Microenvironment Screeningand Development

Materials.

Xeno Free thiol-modified hyaluronan (HA) containing thiol-modifiedcollagen (TMC) (collectively HAF) and polyethylene glycol diacrylate(CL) were purchased from Glycosan BioSystems, Inc. (Salt Lake City,Utah). Human dermal fibroblasts (HDF) were purchased from Invitrogencorporation (Carlsbad, Calif.). mTeSR®1 medium and 10×collagenase/hyaluronidase were purchased from Stem Cell Technologies,Inc. (Vancouver, BC, Canada). Laminin, fibronectin and vitronectin, andsoluble form of basement membrane purified from Engelbreth-Holm-Swann(EHS) tumor containing laminin I, collagen IV, entactin, heparin sulfateproteoglycan were from Trevigen Corp. (VWR Scientific, West Chester,Pa.). Epidermal Growth Factor and Fibroblast Growth Factor were fromCollaborative Research, Inc. (Bedford, Mass.). 6-bromoindirubin-3′-oxime(BIO) and 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridinewere purchased from EMD4 Biosciences Corp (Poland).Indirubin-5-nitro-3′-oxime (INO) (custom synthesis). Prostaglandins J2and E2 were from Cayman Chemicals Corp. (Ann Arbor, Mich.). ValproicAcid (2 mM), colcemid (N-deacetyl-N-Methylcolchicine) were fromSigma-Aldrich (St. Louis Mo.).1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyflethanone.HBr (Pifithrin-α) was from Enzo Life Sciences (San Diego, Calif.).

Cells and Media.

Human embryonic stem cells (hESC, MEL-1; Millipore Corp.; Billerica,Mass.) and human dermal fibroblasts (HDF; Invitrogen; Carlsbad, Calif.)cells were used for screening of conditions. In all cases, afterpreparation, biomaterial microarrays were seeded with 100,000 cells in a100 mm culture dish using fully supplemented mTeSR-1 medium (Stem CellTechnologies; Vancouver, BC, Canada). Cells were also grown on Matrigel(BD Biosciences) coated plates in presence of mTeSR-1 medium as acontrol.

Pig Small Intestine Submucosa (SIS) Extract.

Small intestinal submucosa (SIS) was prepared from porcine intestineobtained from a local meat processing plant. Intestine was rinsed freeof contents, everted and the superficial layers of the mucosa wereremoved by mechanical delamination. The tissue was reverted to itsoriginal orientation and the external muscle layer removed. The preparedSIS tube was split open longitudinally and rinsed extensively in waterto lyse any cells associated with the matrix and to eliminate celldegradation products. Immediately after rinsing, SIS was frozen inliquid nitrogen and stored at −80° C. Frozen tissue was sliced into 1 cmcubes, pulverized under liquid nitrogen with an industrial blender toparticles less than 2 mm² and stored at −80° C. prior to use. SIS powderwas suspended in extraction buffer (4 M guanidine, 2 M urea, 2 M MgCl₂and 2 M NaCl, in 50 mM Tris-HCl, pH 7.4) (25% w/v) containingphenylmethyl sulphonyl fluoride, N-ethylmaleimide, and benzamidine(protease inhibitors; 1 mM each) and vigorously stirred for 24 hours at4° C. The extraction mixture was then centrifuged at 12,000×g for 30minutes at 4° C. and the supernatant collected. The insoluble materialwas washed briefly in the extraction buffer, centrifuged, and the washcombined with the original supernatant. The supernatant was dialyzedextensively in Spectrapor tubing (MWCO 3500, Spectrum MedicalIndustries, Los Angeles, Calif.) against 30 volumes of deionized water(9 changes over 72 hours). The dialysate was centrifuged at 12,000×g toremove any insoluble material and the supernatant was used immediatelyor lyophilized for long term storage.

Fresh Liver Crude Extract.

Freshly isolated liver from porcine was obtained from a local meatprocessing plant. Immediately after isolation the liver was frozen inliquid nitrogen and stored at −80° C. Frozen tissue was sliced into 1 cmcubes, pulverized under liquid nitrogen with an industrial blender toparticles less than 2 mm² and stored at −80° C. prior to use. Usingextraction buffer made of 4 M guanidine, 2 M urea, 2 M MgCl₂ and 2 MNaCl each prepared in 50 mM Tris-HCl, pH 7.4. Liver powder was suspendedin extraction buffers (25% w/v) containing phenylmethyl sulphonylfluoride, N-ethylmaleimide, and benzamidine (protease inhibitors) eachat 1 mM and vigorously stirred for 24 hours at 4° C. The extractionmixture was then centrifuged at 12,000×g for 30 minutes at 4° C. and thesupernatant collected. The insoluble material was washed briefly in theextraction buffer, centrifuged, and the wash combined with the originalsupernatant. The supernatant was dialyzed extensively in Spectraportubing (MWCO 3500, Spectrum Medical Industries, Los Angeles, Calif.)against 30 volumes of deionized water (9 changes over 72 hours). Thedialysate was centrifuged at 12,000×g to remove any insoluble materialand the supernatant was used immediately or lyophilized for long termstorage.

Biomaterial Microarray Slide Preparation.

Epoxy coated glass slides (Corning) were dip coated into 4% (w/v) polyhydroxyethyl methacrylate (pHEMA; Aldrich; Milwaukee, Wis.) solution inethanol and dried for 3 days prior to use. Polymers were purchased fromAldrich, Polysciences (Warrington, Pa.), and Birmingham Polymers(Birmingham, Ala.). Polymers were dissolved to 10% w/v indimethylformamide (DMF), and then mixed in 384 well plates. Monomerswere printed onto the slides using CMP6 or CMP3 pins (TelechemInternational, Sunnyvale, Calif.) with a Pixsys 5500 robot (CartesianTechnologies, Inc.: Ann Arbor, Mich.). To increase the thickness ofpolymer spots, six layers of polymer were printed onto each spot. Thepolymer arrays were then dried at <50 mTorr for at least 7 days. Polymerarrays were sterilized by exposure to UV for 30 min on each side, andthen washed twice with PBS for 30 min and then twice with medium for 30min prior to use.

Combinatorial mixtures (see below) of a) Polymers, b) ECM factors, c)Growth Factors, and d) Crude factors were screened in both cell embedded(3D) and non-embedded (2D) format.

Analysis and Scoring.

After 5 days in culture, each spot was visually examined and scored forcell growth, survival and overall appearance of culture health, andcompared to controls grown on Matrigel using the following relativescoring system:

Numerical Assessment of Culture Growth, Score Survival and CultureHealth 10 Much better than Matrigel control 5 Similar to Matrigelcontrol 1 Much worse than Matrigel control

Healthy hESC had the characteristic, undifferentiated appearance ofcompact cells having clearly defined cell borders. Healthy cultures ofboth hESC and HDF showed evidence of active mitosis. Scores from bothcell type (hESC & HDF) were averaged for each condition.

Polymer Screening.

Individual polymers (Table 1; all from (Sigma-Aldrich; St. Louis, unlessindicated otherwise) were screened initially.

TABLE 1 Polymers Screened A Poly(1,4-butylene adipate) B Poly(ethyleneadipate) C Poly(1,3-propylene succinate) D Poly(1,3-propylene glutarate)E Poly(1,3-propylene adipate) F Poly(D,L-lactide-co-caprolactone)lactide:caprolactone 40:60 G Poly(D,L-lactide-co-caprolactone)lactide:caprolactone 84:16 H Poly(1,4-butylene adipate), diol end-cappedI Poly(ethylene adipate), dihydroxy terminated JPoly(lactide-co-glycolide) lactide:glycolide 50:50, MW_60,000 KPoly(lactide-co-glycolide) lactide:glycolide 50:50, MW_18,000, acidterminated L Poly(lactide) D:L 50:50 MW_25,000 MPoly(lactide-co-glycolide) lactide:glycolide 50:50, MW_65,000 NPoly(lactide-co-glycolide) lactide:glycolide 85:15, MW_60,000 OPoly(lactide-co-glycolide) lactide:glycolide 65:35, MW_58,000 PPoly(lactide-co-glycolide) lactide:glycolide 75:25, MW_100,000 QPoly(lactide-co-glycolide) 1-lactide, lactide:glycolide 70:30, MW_22,000R Poly(lactide-co-glycolide) lactide:glycolide 50:50, MW_100,000 SPoly(lactide) L:DL 60:40, MW_120,000 T Poly(lactide) D:L 50:50,MW_8,000, acid terminated U Poly(lactide-co-glycolide-co-glycol)lactide:glycolide:glycol 53:21:26 MW_80,000 V Poly(ethlyene glycol)MW_300 W Poly(lactide-co-glycolide) lactide:glycolide: 65:35 MW_14,000 XPoly(azelaic anhydride) Y Hyaluronan (Glycosan BioSystems) Z Alginate(Invitrogen) a Acrylamide b Gelatin c Methylcellulose d Agar e Control(Matrigel)

Each single polymer spot contained soluble form of basement membranepurified from Engelbreth-Holm-Swarm (EHS) tumor containing laminin I,collagen IV, entactin; heparin sulfate proteoglycan (EHS basementmembrane; 50 μg/ml). Hyaluronan hydrogel had the highest score from thisscreen, therefore, all other factor screens were performed usinghyaluronan as the hydrogel. The results of the polymer screening areshown in FIG. 1. Values are averaged from experiments with both hESC andHDF cells.

Extracellular Matrix Factor Screening.

Individual extracellular matrix (ECM) factors (Table 2; all fromSigma-Aldrich; St. Louis, unless indicated otherwise) were screened asdescribed above for polymers.

TABLE 2 Extracellular Matrix Factors Tested A Laminin (5 μg/ml) BFibronectin (5 μg/ml) C Vitronectin (6 μg/ml) D Collagen (10 μg/ml) ERGD peptide (40 μg/ml) F IKVAV peptide (50 μg/ml) G Soluble form ofbasement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor(containing laminin I, collagen IV, entactin, heparin sulfateproteoglycan; 50 ug/ml) (EHS basement membrane) H Control (Matrigel)The results of the polymer screening are shown in FIG. 2. Based on thisscreening, laminin, fibronectin, vitronectin and EHS soluble form ofbasement membrane were selected for further study.

Individual Growth Factor Screening.

Individual growth factors (Table 3) were screened as described above forpolymers.

TABLE 3 Growth Factors Tested A Epidermal Growth Factor (EGF; 40 ng/ml)B Fibroblast Growth Factor, (FGF; 220 ng/ml) C Noggin (150 ng/ml) D BDNFE NGF F Insulin G IGF-1 H IGF-2 I PDGF J Control (Matrigel)The results of the growth factor screening are shown in FIG. 3. Based onthis screening, EGF, FGF and Noggin were selected for further study.

Crude Growth Factor Extract Screening.

Crude growth factors (Table 4) were screened as described above forpolymers.

TABLE 4 Crude Factor Extracts Tested A Fresh liver extract (50 μl/ml) BLiver Extract S100 Fresh liver extract S100 fraction (Supernatant ofextract centrifuged at 100,000 × g for 30 minutes) (50 μl/ml) C SIS PigSmall Intestine Submucosa extract (50 μl/ml). D SIS-S100 Pig SmallIntestine Submucosa (SIS) extract, S100 fraction (Supernatant of extractcentrifuged at 100,000 × g for 30 minutes) (50 μl/ml). E Soluble EHS FControl (Matrigel)The results of the crude growth factor extract screening are shown inFIG. 4. Based on this screening, SIS and Soluble EHS were selected forfurther study.

Example 2. Combinatorial Subtractive Optimization of MicroenvironmentNiche Composition

To optimize the microenvironment niche for maintaining hES and HDFcells, a complete medium containing ECM factors, individual growthfactors and crude growth factors as in EXAMPLE 1 that gave a score 3 orhigher were combined to arrive at a Complete Media formulation: laminin(5 μg/ml), fibronectin (5 μg/ml), vitronectin (6 μm/ml); EGF; 40 ng/ml);FGF (220 ng/ml); Noggin (150 ng/ml) SIS (500 ml); and EHS (50 μm/ml) inmTeSR-1. To confirm the benefit of each component of the Complete Media,each factor a combinatorial subtractive screening was performed.Briefly, cells were grown on hyaluronan hydrogel polymer as describedabove in EXAMPLE 1 in either 2D (cells on the surface of polymerizedhydrogel) or 3D (cells are embedded in the hydrogel) in Complete Mediaor Complete Media missing one of the components, as indicated below inTable 5.

TABLE 5 Components for Combinatorial Subtractive Screening A CompleteMedia B Complete minus SIS C Complete minus Growth Factors D Completeminus ECM E Complete minus laminin F Complete minus fibronectin GComplete minus vitronectin H Complete minus EGF I Complete minus FGF JComplete minus Noggin K Complete minus EHS L Control (Matrigel)

The results of the combinatorial subtractive screening are shown in FIG.5. The results of this screening confirmed that the optimal growthresults were obtained when cells were grown in the ES cellmicroenvironment niche provided by 3D culture with Complete Media. Itshould be noted that each of the combinations were tested in both 2D and3D format and the 3D format yielded consistently higher scores.

Example 3: Chemical Induction of Pluripotency

ViPS strategies have focused on induction of pluripotency in somaticcells by introducing exogenous polynucleotides into the somatic cell viaviral expression vectors. The polynucleotide express a discrete set offactors (“pluripotency factors”) believed responsible for inducingpluripotency. We reasoned that compounds known to stimulate expressionof these pluripotency factors could be substituted for the exogenouspolynucleotides.

Reported studies indicated that one set of pluripotency factors that wassufficient for induction of pluripotency was OCT4, Sox2, Nanog, andKlf4. To test the possibility of chemical induction of pluripotency inhuman dermal fibroblasts, well known compounds affecting thesepluripotency factors were substituted for expression of the pluripotencyfactors from viral expression vectors. Candidate compounds includedthose that affect the Wnt pathway because activation of this pathway hadbeen reported to be involved in OCT 3/4, Sox-2, and Nanog activation.The initial compounds tested included: BIO (6-bromoindirubin-3′-oxime),a specific pharmacological inhibitor of glycogen synthase kinase knownto stimulate Wnt signaling and maintain pluripotency in human and mouseembryonic stem cells (Sato et al., Nat. Med. (2004) 10:55-63);prostaglandin E2 (PGE2), reported to be an activator of Wnt pathway via“cross talk” at the beta-catenin signal level; valproic acid, a histonedeacetylase (HDAC) inhibitor, and Wnt pathway activator (Bug et al.(2005) Cancer Res 65:2537-41). Valproic acid's influence on HDACs wasalso suggested to part in eliminating the need for KLF4 gene activation(Evans et al. (2007) J. of Biol. Chem. 282:33994-34028); andprostaglandin J2 (PGJ2), known to increase endogenous KLF4 geneexpression levels (Chen & Tseng (2005) Mol. Pharmacol. 68:1203-13).These compounds were chosen for further experimentation since theirmechanism of activity was previously determined and they appeared toactivate the pluripotency signal transduction pathway (e.g. Wntpathway).

Compounds were added to HDF cells grown in 3D ES cell microenvironmentniche cultures (prepared as described above in EXAMPLES 1 and 2), toevaluate whether they would rescue iPS phenotype in the absence ofindividual pluripotency factors introduced retrovirally. ViPS cells,induced with lentiviral transduction of pluripotency factor genes (OCT4,Sox2, Nanog, Klf4) as described by Takahashi et al. ((2007), Cell131:1-12). Data is presented as the number of colonies with iPSmorphology observed per 100,000 host cells. The results summarized inFIG. 6 provide preliminary evidence in support of the hypothesis thatcompounds including BIO, PGE2, valproic acid and PGJ2, could be used toreplace virally transduced pluripotency factors in induction ofpluripotent status in somatic cells. Surprisingly, a cocktail of thesefour compounds only resulted in the rescue of the phenotype of ViPScells using small molecules only.

Stem cell self renewal optimized 3-D hydrogel ES microenvironment nicheconditions (as described in EXAMPLEs 1 and 2) were found necessary forchemical induction of iPS state. Pluripotency was not observed whenstandard tissue culture plates or those coated with Matrigel ormethylcellulose were substituted for the 3D microenvironment conditions.

Example 4: Screening for Compounds Capable of Reprogramming of HDF Cells

To screen for additional and/or more effective compounds capable ofreprogramming somatic cells to pluripotency, various compounds weretested for their effect on somatic HDF cells in optimized 3Dmicronenvironment niche culture. For the initial screen, the ability oftest compounds to affect genes known to be expressed in pluripotentcells was measured. HDF cells and biomaterial microarrays were preparedas follows.

Induction and Screening.

Replicate microspots were prepared in Complete Media and transfectedwith reporter constructs for genes critically expressed in pluripotentcells. The reporter constructs contained promoter regions of one of thegenes listed in Table 6 operably linked to a green fluorescent protein(GFP) coding region in pGlow TOPO TA (Invitrogen).

TABLE 6 Promoters for Pluripotency Gene Expression Reporter ConstructsGene Description GenBank Identifier OCT3/4 POU class 5 homeobox 1GI:11602730 (POU5F1) SOX2 sex determining region Y GI:215820640 box2LIN28 lin28 homolog GI:224589800 KLF4 Kruppel like factor 4 (gut)GI:224589821

Briefly, a transient transfection mixture containing Lipofectamine 2000(0.4 μl/24 μl total hyaluronan volume, Invitrogen) and promoter-GreenFluorescent Protein (GFP) construct in (500 ng) was added to eachmicrospot.

Compound Libraries. An in-house library of compounds (3600 compounds)was assembled by combining commercially available compound libraries andpreviously known activators of self renewal/stemness as well aspotential self-renewal modulators of unknown mechanism of action. Thelibraries included compounds from: MicroSource library, which contained2000 biologically active and structurally diverse compounds from knowndrugs, experimental bioactives, and pure natural products; including acollection of 720 natural products and their derivatives, a range ofsimple and complex oxygen-containing heterocycles, alkaloids,sesquiterpenes, diterpenes, pentacyclic triterpenes, sterols, and manyother diverse representatives; and the Prestwick Chemical Library (seethe world wide web at prestwickchemical “dot” fr), a collection of 880high-purity chemical compounds selected for structural diversity andrepresenting drug classes with a broad spectrum of therapeutic uses.More than 85% of its compounds are marketed drugs. Notably, thelibraries included 6-bromoindirubin-3′-oxime (BIO), valproic acid,prostaglandin E2 (PGE2), and prostaglandin J2 (PGJ2).

Many compounds in the library could be assigned to one of 14 highinterest categories based on known targets and pathways affected, andreported outcomes, as shown in Table 7.

TABLE 7 Categories of Compounds Screened Compound Outcome CategoryTarget Pathway (Promotes) 1 GSK-3 Wnt Self Renewal 2 GSK-3 WntDifferentiation 3 TBD Wnt Self Renewal 4 TBD Wnt Differentiation 5Smoothened Hedgehog Anti-Proliferation 6 TBD Hedgehog Anti-Proliferation7 TBD Hedgehog Differentiation 8 TBD NF-kB Self Renewal 9 TBD NF-kBDifferentiation 10 Cox-1 PGE-2 Anti-Proliferation 11 Cox-1 PGE-2Anti-Proliferation 12 PKC PKC Apoptosis, Necrosis 13 TBD TBD SelfRenewal 14 TBD TBD Differentiation

Library screening was performed by including dilution series of eachcompound in mTeSR-1 medium, to HDF cells embedded in 3D hyaluronanhydrogel microenvironment niche culture optimized as described above inEXAMPLES 1 and 2.

After 4 days of incubation with target compounds, relative fluorescencefrom expression of the GFP constructs was recorded using a fluorescentmicroplate reader. Values were normalized to reflect percentfluorescence of control (ViPS) cells. A sampling data of thishigh-throughput screen using the compounds listed in Table 8 is shown inFIG. 7.

TABLE 8 Exemplary Compounds Screened A D1-234 B D7-874 C D2-435 D D7-988E D2-2334 F D9-6452 G D16-34675 H D3-8976 I D12-2234 J D2-6542 K D6-982L D21-8765 M D23-1276 N D6-987 O D10-8976 P D-2217 Q D9-9811 R D14-3245S D9-7765

Compounds that activated expression of one or more of the targetpromoters were scored as positive. A set of positive compounds from thescreen shown in FIG. 6 were: D9-6452: 6-bromoindirubin-3′-oxime (BIO);D10-8976: Valproic Acid; D3-8976: Indirubin-5-nitro-3′-oxime (INO);D21-8765:2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine;D12-2234:1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanoneHBr, (Pifithrin-alpha); D9-9811: Prostaglandin J2; D2-6542:Prostaglandin E2.

Positive compound hits were selected, rescreened in triplicate and thebest candidates chosen on the basis of highest level of promoteractivation for one or more target promoters tested. Through multiplerounds of repeat experimentations with a broad range of compoundconcentrations, optimum concentration for each of the selected compoundswas empirically determined. The optimized concentrations of sevencompounds are summarized in Table 9.

TABLE 9 Optimized Chemical Induction Component Concentrations ChemicalInduction Component Optimized Concentration 6-bromoindirubin-3′-oxime(BIO) 4 μM Indirubin-5-nitro-3′-oxime (INO) 4 μM Valproic Acid 2 mMD21-8765: 2-(3-(6-Methylpyridin-2-yl)- 25 mM1H-pyrazol-4-yl)-1,5-naphthyridine1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro- 30 μM2-imino-3(2H)-benzothiazolyl)ethanone HBr, Pifithrin-alpha ProstaglandinJ2 10 μM Prostaglandin E2 10 μM

Example 6: Optimization of Chemical Induction of Pluripotency

FIG. 8 shows a sampling of data generated using compounds that increasedexpression of promoters from pluripotency genes in the promoter reporterassay screen as described in EXAMPLE 5. Probable candidates were chosenbased on their level of influence on one or more promoters. Alsopositive hits that had a known mechanism of activity on related signaltransduction pathways were favored. Each compound cocktail was includedin a long term induction protocol of CiPSC from HDF. In all cases aserial concentration range of each member of each compound cocktail wereexamined to empirically determine optimal compound/cocktailconcentrations. These optimum concentrations were then used incomparative experiments, result of which are shown in this figure.Number of CiPSC colonies generated per 100000 starting number of HDFcells (Y-axis) was the experiment's end point analysis.

Control experiments were ViPS derivation with lentiviral vectors toinduce pluripotency (A-J). K-W show attempts to use small moleculeinducer cocktails to rescue the iPS phenotype in the absence of geneticinduction. Most successful cocktail is that shown in R. This cocktailwas used for further validation studies. In further experimentaloptimization studies it was determined that synchronization of cellsincreased efficiency of induction. For example treatment with Colcemid,resulted in increased efficiency (see FIG. 7, X)

Members of this cocktail are:

-   -   D9-6452: 6-bromoindirubin-3′-oxime (BIO). Previously known        mechanism: Wnt pathway activation.    -   D10-8976: Indirubin-5-nitro-3′-oxime (INO). Previously known        mechanism: Wnt pathway activation.    -   D3-8976: Valproic Acid. Previously known mechanism: HDAC        inhibitor, Wnt pathway activator, increases KLF4 expression.    -   D21-8765:        2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.        Previously known mechanism: inhibitor of TGFbeta pathway.    -   D12-2234:        1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanone        HBr, Pifithrin-alpha. Previously known mechanism: p53        inhibition.    -   D9-9811: Prostaglandin J2. Previously known mechanism: Increase        endogenous KLF4 levels.    -   D2-6542: Prostaglandin E2. Wnt pathway activator, “crosstalk” at        the Beta-Catenin signal level.

Based on these findings, we concluded that in order to activate thepluripotency pathway and induce reprogramming of somatic cells thefollowing types of intercellular signal modulation are required by theCiPS small molecule cocktails:

1) Molecules that activate Wnt pathway and display characteristicpluripotency gene activation shown.

-   -   2) Molecules that activate Cyclooxygenase pathway and its cross        talk with wnt pathway and display characteristic pluripotency        gene activation shown.    -   3) Histone Deacetylases that activate the pluripotency pathway        and display characteristic pluripotency gene activation shown.    -   4) Molecules that increase Sox-2 expression and display        characteristic pluripotency gene activation shown.    -   5) Molecules that increase Nanog expression and display        characteristic pluripotency gene expression.    -   6) Molecules that inhibit p53 and related pathways and display        characteristic pluripotency gene activation shown.    -   7) Synchronization of cells prior to or during induction of        CiPSC improves efficiency.

Example 7: Induction of Pluripotency in Human Dermal Fibroblasts

Preparation of 3D Format Cultures. HA and CL were prepared under sterileconditions according manufacturer's directions. HDF were prepared. 8.0mL of HA was mixed with 8.0 mL of TMC. The following additives wereadded to the mixture in a total volume of ≤4 mL to the indicated finalconcentrations: laminin (5 μg/ml); fibronectin (5 μg/ml); vitronectin (6μg/ml); Epidermal Growth Factor (40 ng/ml); Fibroblast Growth Factor(220 ng/ml); Noggin (150 ng/ml); Pig Small Intestine Submucosa (SIS)extract, (50 μl/ml); soluble form of basement membrane purified fromEngelbreth-Holm-Swarm (EHS) tumor containing laminin I, collagen IV,entactin, heparin sulfate proteoglycan. (50 μg/ml). The mixture was theninverted and vortexed for 10 minutes at 2° C. This final solution wasreferred to as complete HAF (HAFC).

Human dermal fibroblasts (HDF) 0.2 mL cells, were added to 2 mL HAFC andthe mixture gently pipeted to mix. CL (0.5 mL) of was then added to thecell mixture to form hydrogels, giving a final cell density of 50cells/ml and 1 ml was dipesensed per well of a 24 culture plates. Theplates were then incubated at 37° C. incubator with 5% CO2 for 1 hour toallow HAFC to gel. After gelling, 1.8 mL of mTeSR-1 culture media wasadded to each well, and the cells incubated at 37° C. incubator with 5%CO2 with a change of mTeSR-1 media every two days. During the firstweek, the media included 0.01 μg/ml of colcemid.

Induction.

Induction was initiated 1 week after culture and continued for threeweeks. The following Inducer Drug Cocktail (IDC) was included in mTeSRmedium: 6-bromoindirubin-3′-oxime (4 μM); indirubin-5-nitro-3′-oxime (4μM); valproic vcid (2 mM);2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (25 mM),1-(4-methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanoneHBr, pifithrin-alpha (30 μM); prostaglandin J2 (10 μM), andprostaglandin E2 (10 μM). Cell culture was continued for a total of 32days after initial plating.

Cells were recovered by adding using 500 μL collagenase/hyaluronidasesolution (diluted 1:2 in mTeSR-1 media) per 100 μL of hydrogel withgentle shaking overnight at 37° C. The cells were recovered bycentrifugation at 1500 rpm for five minutes and washed with PBS.Recovered cells were resuspended in 0.5 mL of mTeSR media withoutchemical inducers.

CiPS cells were subcloned and propagated on the surface of HAFC (2-DCulture) according to standard tissue culture protocols.

Example 8: Validation of Cell Reprogramming

Pluripotency Gene Expression.

RT-PCR was performed to measure expression of pluripotency associatedgenes. Briefly, total cellular RNA was extracted from 5×10⁶ CiPS cellsusing a RNeasy Protect Mini kit (Qiagen; Valencia, Calif.), according tothe manufacturer's instructions, and reverse transcribed using aSuperScript III First-Strand Synthesis System RT-PCR (Invitrogen). ThecDNA was amplified by PCR using Accuprime Taq DNA polymerase system(Invitrogen).

Primers used for analysis of endogenous CiPS gene expression are shownin Table 10.

TABLE 10 Primers for Gene Expression Analysis Primer SEQ ID Gene NamePrimer Sequence NO hNANOG hNANOG- 5′-GCAGAAGGCCTCAGCACCTA- SEQ ID  F 3′NO: 1 hNANOG- 5′-AGGTTCCCAGTCGGGTTCA- SEQ ID  R 3′ NO: 2 hOCT4 hOCT4-F5′-GCTCGAGAAGGATGTGGTCC- SEQ ID  3′ NO: 3 hOCT4-R5′-CGTTGTGCATAGTCGCTGCT- SEQ ID  3′ NO: 4 hSOX2 hSOX2-F5′-CACTGCCCCTCTCACACATG- SEQ ID  3′ NO: 5 hSOX2-R5′-TCCCATTTCCCTCGTTTTTCT- SEQ ID  3′ NO: 6 hKLF4 hKLF4-F 5′- SEQ ID GCGAACTCACACAGGCGAGAAACC- NO: 7 3′ hKLF4-R 5′- SEQ ID TCGCTTCCTCTTCCTCCGACACA- NO: 8 3′ hREX-1 hREX-1- 5′-GGCTTCCCTGACAGATACC-SEQ ID  F 3′ NO: 9 hREX-1- 5′ CCTTCGAACGTGCACTGATA  SEQ ID  R 3′ NO: 10hGAPDH- hGAPDG- 5′ ACCACAGTCCATGCCATCAC  SEQ ID  R F 3′ NO: 15 hGAPDH-5′ TCCACCACCCTGTTGCTGTA  SEQ ID  R 3 NO: 16

PCR products were separated by electrophoresis on a 2% agarose gel,stained with ethidium bromide and visualized by UV illumination. Theresults of this analysis are shown in FIG. 9.

Pluripotency Biomarker Expression.

Cells were fixed in 4% paraformaldehyde in PBS and immunostainedaccording to standard protocols using the following primary antibodies:SSEA4 (mouse monoclonal, Developmental Studies Hybridoma Bank); Tra1-60, (mouse monoclonal, Chemicon International); hSOX2 (goatpolyclonal, R&D Systems); Oct-3/4 (mouse monoclonal, Santa CruzBiotechnology); hNANOG (goat polyclonal R&D Systems); appropriateMolecular Probes Alexa Fluor® dye conjugated secondary antibodies(Invitrogen) were used. The results of this analysis are summarized inTable 11.

TABLE 11 Biomarker Expression of hES Cells and CiP Cells Marker hESCCiPC SSEA-1 — — SSEA-3 + + SSEA-4 + + TRA-1-60 + + TRA-1-81 + + OCT4 + +

Mouse Teratoma Analysis.

Approximately 1-3×10⁶ CiPSC were injected subcutaneously into the testesof nude mice SCID mice (Jackson labs) anesthetized with isoflurane. Fiveto 6 weeks after injection, teratomas formed and were dissected, fixedovernight in 10% buffered formalin phosphate and embedded in paraffin.Sections were stained with haematoxylin and eosin for further analysis.Tissue sections were analyzed by light microscopy as shown in FIG. 10.Regions representing the following tissues were observed and markedproving presence of tissues from all three main somatic germ layers(Ectoderm, Mesoderm, Endoderm). P: Pigmented Epithelium, EP: EctodermalEpithelium, B: Bone (Mesoderm), CA: Cartilage (Mesoderm), EN-EP:Endodermal Epithelium, MU: Striated Muscle (Mesoderm), A: AdiposeTissue, N: Neural Tissue (Ectoderm).

Methylation Analysis

DNA was isolated from CiPSC and hESC and treated with bisulfite usingthe EpiTect Bisulfite Kit (Qiagen) according to manufacturer'sinstructions. Amplified products were purified using gel filtrationcolumns (Qiagen), cloned into the pCR2.1-TOPO vector (Invitrogen), andsequenced with M13 forward and reverse primers. Unmethylated ormethylated CpGs were determined, as shown in FIG. 11. Open and closedcircles indicate unmethylated and methylated CpG, respectively. Numbers(right) indicate CpG locations. Percentages of CpG methylation areshown. Percent methylation values for Oct 4 and Nanog promoter regionsare similar between hESC and CiPSC.

1-14. (canceled)
 15. A composition for reprogramming cells, thecomposition comprising: a polymer hydrogel; valproic acid; and at leastone reprogramming agent.
 16. The composition of claim 15, wherein thecomposition is capable of reprogramming at least one somatic cell topluripotency.
 17. The composition of claim 16, wherein the at least onepluripotent cell is a human stem cell.
 18. The composition of claim 17,wherein the human stem cell is an induced pluripotent stem cell.
 19. Thecomposition of claim 16, wherein the at least one pluripotent cell is anembryonic stem cell.
 20. The composition of claim 19, wherein theembryonic stem cell is a human embryonic stem cell.
 21. The compositionof claim 15, wherein the polymer hydrogel further comprises a polyethercross linker.
 22. The composition of claim 21, wherein the polyethercross linker is polyethylene glycol diacrylate.
 23. The composition ofclaim 15, wherein the composition further comprises hyaluronan and/orthiol-modified collagen.
 24. The composition of claim 23, wherein thehyaluronan is thiol-modified.
 25. The composition of claim 15, whereinthe polymer hydrogel further comprises at least one component selectedfrom the group consisting of laminin, fibronectin, vitronectin,epidermal growth factor, fibroblast growth factor, Noggin, SIS, and EHSbasement membrane.
 26. The composition of claim 15, wherein the at leastone reprogramming agent comprises small molecules, proteins, antibodies,DNA vectors, miRNA, oligonucleotides, synthetic RNA molecules andsynthetic DNA.
 27. The composition of claim 26, wherein the at least onereprogramming agent further comprises at least one component selectedfrom the group consisting of 6-bromoindirubin-3′-oxime (BIO);indirubin-5-nitro-3′-oxime (INO);2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-napthyridine;1-4(-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanone HBr (Pifithrin-α); prostaglandin J2; and prostaglandin E2. 28.The composition of claim 27, wherein the at least one reprogrammingagent activates at least one signal transduction pathway.
 29. Thecomposition of claim 28, wherein the at least one signal transductionpathway is selected from the group consisting of Wnt; Hedgehog;Cyclooxygenase; p53; NF-κB; PKC; and PGE-2 pathways.
 30. A compositionfor reprogramming cells, the composition comprising: a polymer hydrogel;valproic acid; and at least one signal transduction pathway modulator.31. The composition of claim 30, wherein the composition is capable ofreprogramming at least one somatic cell to pluripotency.
 32. Thecomposition of claim 31, wherein the at least one pluripotent cell is asomatic stem cell.
 33. The composition of claim 30, wherein the at leastone signal transduction pathway modulator is a growth factor.
 34. Thecomposition of claim 33, wherein the at least one growth factor isselected from the group consisting of epidermal growth factor;fibroblast growth factor; Noggin; BDNF; NGF; Insulin; IGF-1; IGF-2; andPDGF.